A Lipopolysaccharide-Binding Domain of the Campylobacter fetus S-Layer Protein Resides within the

J OURNAL OF B ACTERIOLOGY,Apr.1995,p.1734–1741Vol.177,No.7 0021-9193/95/$04.00ϩ0Copyright᭧1995,American Society for MicrobiologyA Lipopolysaccharide-Binding Domain of the Campylobacter fetusS-Layer Protein Resides within the Conserved N Terminusof a Family of Silent and Divergent HomologsJOEL DWORKIN,1MURALI K.R.TUMMURU,1AND MARTIN J.BLASER1,2*Division of Infectious Diseases,Department of Medicine and Department of Microbiology and Immunology, Vanderbilt University School of Medicine,Nashville,Tennessee37232,1and Medical Service,Department of Veterans Affairs Medical Center,Nashville,Tennessee372122Received1November1994/Accepted25January1995Campylobacter fetus cells can produce multiple S-layer proteins ranging from97to149kDa,with a singleform predominating in cultured cells.We have cloned,sequenced,and expressed in Escherichia coli a sapAhomolog,sapA2,which encodes a full-length1,109-amino-acid(112-kDa)S-layer parison with thetwo previously cloned sapA homologs has demonstrated two regions of identity,approximately70bp before theopen reading frame(ORF)and proceeding550bp into the ORF and immediately downstream of the ORF.Theentire genome contains eight copies of each of these conserved regions.Southern analyses has demonstratedthat sapA2existed as a complete copy within the genome in all strains examined,although Northern(RNA)analysis has demonstrated that sapA2was not expressed in the C.fetus strain from which it was cloned.FurtherSouthern analyses revealed increasing sapA diversity as probes increasingly3؅within the ORF were used.Pulsed-field gel electrophoresis and then Southern blotting with the conserved N-terminal region of the sapAhomologs as a probe showed that these genes were tightly clustered on the chromosome.Deletion mutagenesisrevealed that the S-layer protein bound serospecifically to the C.fetus lipopolysaccharide via its conservedN-terminal region.These data indicated that the S-layer proteins shared functional activity in the conservedN terminus but diverged in a semiconservative manner for the remainder of the molecule.Variation in S-layerprotein expression may involve rearrangement of complete gene copies from a single large locus containingmultiple sapA homologs.Campylobacter fetus is a spiral,microaerophilic gram-nega-tive bacterium that causes infertility and infectious abortion in sheep and cattle and extraintestinal infections in humans(16, 23,43,45).There are two serotypes of C.fetus(A and B),a property based on their lipopolysaccharide(LPS)composition (34).As with many other bacterial species(2),C.fetus pos-sesses an outermost crystalline surface layer of regular closely packed protein subunits(S-layer proteins).The S-layer pro-teins of C.fetus have been shown to be critical in resistance to host immune defenses(5–7,32,51).C.fetus strains possessing S-layer proteins(Sϩ)but not spontaneous mutants or chemi-cally treated strains lacking S-layers(SϪ)are resistant to C3b binding and phagocytosis by polymorphonuclear leukocytes(5, 7).The C.fetus S-layer proteins must share relatively conserved features,as dictated by requirements for crystalline structure and binding to divalent cations(19,22,53)and as demon-strated by amino-terminal sequencing(33)and cross-reactivity with polyclonal antisera(6,18,49).In addition,S-layer pro-teins bind exclusively to the LPS of homologous(type A or B) cells,which is a property that is localized to the N-terminal half of the molecule(53).Cloning of two genes(sapA and sapA1) encoding97-kDa forms of S-layer proteins has demonstrated that the S-layer proteins share identical amino termini extend-ing for219amino acid residues but diverge for the remainder of the molecule(4,47).A characteristic of the C.fetus surface layer is that a single bacterial strain may produce one of three different S-layer proteins ranging from97to149kDa,with one form predom-inating in mixed cultures(8,18,22,33).C.fetus strains may shift the size of the S-layer protein being produced,resulting in a shift in antigenicity and possible evasion of host immune defenses(8,15,18,19,47,49,50).However,the mechanisms by which such changes occur are unknown.Southern blot anal-ysis using the conserved amino-terminal coding region as a probe demonstrates that multiple(8)sapA homologs exist within the C.fetus genome(46);however,it is unknown whether size variation involves reassortment of complete(si-lent)gene copies or assemblage of partial coding gene seg-ments(47).To address this question,we cloned a gene from strain82-40 LP3,which expresses an S-layer protein with an apparent mo-lecular mass of127kDa,as estimated by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis(SDS-PAGE).Analy-sis of this gene provided information as to whether larger S-layer proteins are due to longer open reading frames(ORFs) or reflect posttranslational modification.Further analyses per-taining to the organization of the sapA homologs,the conser-vation of sapA homolog sequences,and the domains involved in specific LPS binding by the gene product also were per-formed.MATERIALS AND METHODSBacterial strains and culture condition.Six C.fetus subspecies fetus strains,of either Sϩor SϪphenotype and LPS serotype A or B,were used in this study (Table1).C.fetus23D was isolated from the vagina of a cow in New York before 1970;C.fetus82-40LP was isolated from the blood of a human in Pittsburgh,Pa., in1981.Stock cultures were kept atϪ70ЊC in brucella broth(BBL Microbiology Systems,Cockeysville,Md.)supplemented with15%glycerol.For study,cultures were grown in a microaerobic atmosphere(5%oxygen,10%carbon dioxide,85%*Corresponding author.Mailing address:Vanderbilt University School of Medicine,Division of Infectious Diseases,A-3310Medical Center North Nashville,TN37232-2605.Phone:(615)322-2035.Fax: (615)343-61601734nitrogen)at37ЊC on brucella agar plates as described previously(33).The Escherichia coli strains that were used in this study,including DH5␣and XL1-blue(Stratagene,La Jolla,Calif.),were grown in LB medium or on L plates (39).Chemicals and enzymes.Isopropyl-␤-D-thiogalactopyranoside(IPTG)and 5-bromo-4-chloro-3-indolyl-␤-galatoside(X-Gal)was purchased from Jersey Lab Supply(Livingston,N.J.)and were used at50and30␮g/ml,respectively.Re-striction enzymes,T4DNA ligase,and E.coli DNA polymerase large(Klenow) fragment were from Promega and U.S.Biochemicals(Cleveland,Ohio).Pro-teinase K was from Sigma Chemical Co.(St.Louis,Mo.).[␣-32P]dATP(650 Ci/mmol)was from ICN Radiochemicals(Irvine,Calif.).Genetic techniques and DNA sequencing analysis.Chromosomal DNA was prepared as described previously(41).Plasmids were isolated by the procedure described by Birnboim and Doly(3).All other standard molecular genetic tech-niques were used,as described elsewhere(39).To sequence plasmid pMJ1, which harbors the complete ORF for sapA2,a series of200-bp exonuclease III nested deletions were created from either direction of the insert using the Erase-a-base system(Promega,Madison,Wis.).The nucleotide sequence was determined unambiguously on both strands by using these nested deletions as double-stranded DNA templates and the dideoxy chain termination procedure as described previously(40).Primers common to the vector sequenceflanking the insert and additional primers required to complete the sequencing were synthesized at the Vanderbilt University DNA Core Facility with a Milligen7500 DNA synthesizer.Nucleotide sequences were compiled and analyzed with the aid of the DNA Strider program(CEA,Gif-Sur-Yvette,France).Amino acid sequence analysis.Predicted primary amino acid sequences were compared by using the Genetics Computer Group sequence analysis programs (University of Wisconsin,Madison,Wis.)with the Gap algorithm(31),with a gap weight of3and a length weight of1.Construction of a C.fetus genomic library.Partial Alu I digests of chromosomal DNA from C.fetus82-40LP3containing fragments of3to10kb were ligated to Eco RI linkers,digested with Eco RI,and ligated into pBluescript harbored within the vector␭ZapII(Stratagene).Packaged material was plated onto XL1-blue and yielded9ϫ105PFU;99%of the plaques were white on plates containing X-Gal and IPTG,indicating insertional inactivation of the vector␤-galactosidase by foreign DNA fragments.The library was amplified on XL1-blue to obtain a higher titer stock(109PFU/ml).Detection and purification of recombinant clones.The amplified␭ZapII bank was screened for S-layer protein expression with an absorbed high-titer rabbit antiserum to the purified97-kDa S-layer protein from C.fetus82-40LP(33). More than10signals per1,000plaques were detected.After20plaques were repicked and reprobed,15recombinants were selected,and single isolated plaques from each were picked and amplified for further study.The plasmids harbored within these phagemids were excised in vivo as detailed by the manu-facturer(Stratagene).Characterization of the recombinant proteins.To characterize further these clones,XL1-blue cells infected with the recombinant phagemid were induced with IPTG,and the lysates were recovered.The lysates were analyzed by SDS-PAGE,as described elsewhere(25).Immunoblotting was performed as de-tailed elsewhere(32)by using a1:5,000dilution of antiserum to the97-kDa S-layer protein from C.fetus82-40LP(33)and goat anti-rabbit immunoglobulin alkaline phosphatase conjugate as the second antibody.The plasmids harbored within the␭ZapII phagemid that encoded S-layer proteins of97kDa or larger were excised in vivo,as described by the manufacturer,and were used for further study.Southern hybridizations. C.fetus chromosomal DNA was digested with Hae III,electrophoresed,transfered to nylon membranes,and hybridized exactly as described elsewhere(46).The probes were gel-purified DNA fragments from pMJ1or PCR products and were radiolabeled by primer extension with random hexameric oligonucleotides(21).Thefilters were washed successively three times for30min each in2ϫSSC(1ϫSSC is0.15M NaCl plus0.015M sodium citrate), 1ϫSSC,and0.5ϫSSC at65ЊC and were exposed to XAR-2X-rayfilm.RNA isolation and Northern(RNA)hybridization analysis.For RNA isola-tion,C.fetus strains were grown overnight on brucella plates,cells were har-vested,and RNA was recovered by one round of hot phenol extraction as described previously(1).The aqueous phase was reextracted once with phenol-chloroform that had been saturated with0.02M sodium acetate(pH5.2)and twice with chloroform,and then the RNA was precipitated in3volumes of absolute ethanol atϪ70ЊC for30min.For Northern hybridization,RNA samples (10␮g)were subjected to electrophoresis on formaldehyde-agarose gels and then transferred to nylon membranes(39).Northern blots then were probed in the presence of50%formamide(39).PFGE.Overnight cultures of C.fetus23D,23B,82-40LP,82-40HP,and82-40 LP3were embedded in agar,lysed,and stored in0.05M EDTA at4ЊC as described elsewhere(38).DNA was digested with Sal I,Sac II,Mlu I,and Bgl I also as described elsewhere(38).Bacteriophage␭(48.5-kb)concatemers were used as size standards.The contour-clamped homogeneous electricfield(CHEF) system(Bio-Rad,Melville,N.Y.)of pulsed-field gel electrophoresis(PFGE)was used to separate DNA fragments in1%agarose gels.The gels were subjected to electrophoresis for24h at8ЊC at175V with pulse times of10to25s and for a further3h at175V with a pulse time of0.5to1.0s or for20h with a pulse time of1to12s in0.05M Tris-borate-EDTA buffer.After electrophoresis,the gels were stained with ethidium bromide,and the DNA fragments were visualized with a UV transilluminator to verify proper digestion.The gels then were trans-ferred to nylon membranes and Southern hybridization was performed as de-scribed above.In vitro reattachment of recombinant S-layer protein to the C.fetus cell surface.The C.fetus SϪstrains,83-88(serotype B)and23B(serotype A),were used as templates for reattachment of recombinant proteins.Cells were har-vested from plate culture and washed twice with C buffer(1mM HEPES [N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid;pH7.5],20mM CaCl2), pelleted,and stored atϪ20ЊC until later use.Recombinant C.fetus protein products were recovered from sonicated recombinant E.coli cultures suspended in C buffer and centrifuged to remove cellular membranes.Protein quantities were determined using the bicinchoninic acid protein assay kit(Pierce,Rockford, Ill.).Approximately10␮g of lysate from recombinant E.coli strains was then mixed with100␮g of C.fetus cells(ϳ2␮l per cell pellet)in30␮l of C buffer,and the mixture was incubated for1h at37ЊC.Following incubation,cells were washed free of unbound recombinant S-layer protein by washing and pelleting successively in1ml of C buffer.Reattachment then was assessed by immunoblot analysis with anti-97-kDa protein following SDS-PAGE,as previously described (53).RESULTSCloning of C.fetus sapA2homolog.Our initial goal was to determine whether the genes encoding S-layer proteins larger than97kDa exist as complete copies within the genome or whether they are constructed by assembly of partial gene cas-settes similar to that seen in other pathogenic organisms(9,17, 29,42,52).For this purpose,we constructed a␭ZapII library containing Alu I-digested chromosomal fragments from strain 82-40LP3,a mixed culture which predominantly expresses a 127-kDa S-layer protein(Table1).The library was screened with immune serum to the97-kDa S-layer protein(33),and FIG.1.Immunoblot analysis of native and recombinant C.fetus S-layer pro-teins with polyclonal antiserum to the97-kDa S-layer protein from strain82-40 nes:A,whole-cell lysate of C.fetus82-40LP3;B,whole-cell lysate of C. fetus23D;C to F,whole-cell lysates from E.coli recombinant clones pMJ110, pMJ100,pMJ2,and pMJ1,respectively.Molecular masses(in kilodaltons)refer to the major S-layer proteins of C.fetus.TABLE1.C.fetus subspecies fetus strains used in this studyStrain OriginPredominateS-layer protein a(kDa)LPS type b84-32(23D)Wild type97A84-54(23B)Spontaneous mutant—c A82-40LP Wild type97A82-40HP Spontaneous mutant—A82-40LP3Spontaneous mutant97,127A83-88Wild type—Ba Major surface array protein expressed by each strain.b LPS types as previously described(34).c—,no S-layer protein expressed.V OL.177,1995LPS-BINDING DOMAIN OF C.FETUS S-LAYER PROTEIN1735immunoreactive plaques were purified and screened for the size of the expressed protein by immunoblotting.Four clones expressed immunoreactive proteins ofՆ97kDa,and a plasmid excised from one of these,pMJ1,expressed a127-kDa form in E.coli,which is referred to as the sapA2product(Fig.1).Nucleotide sequence of sapA2and comparison of sapA ho-mologs.Nested deletions using exonuclease III were made in both directions of pMJ1relative to the insert,and the insert was sequenced on both strands using common vector primers. Figure2shows the4,132-nucleotide and deduced amino acid sequence of sapA2and itsflanking regions.Only a single ORF greater than500bp was found in any of the six possible frames. This3,327-bp ORF encoded a polypeptide of1,109amino acids with a predicted molecular mass of111.8kDa and an apparent molecular mass of127kDa,as estimated by SDS-PAGE(Fig.1).Searches of the protein and gene banks failed to reveal any similarity to other known proteins except other S-layer proteins of C.fetus(4,47).Sequence data(Fig.2) revealed that the region upstream of the sapA2ORF whichwasFIG.2.The nucleotide sequence of pMJ1,including the deduced amino acidsequence of sapA2.The DNA sequence was determined in both directions asdescribed in Materials and Methods.Nucleotides are numbered from thefirstletter of the initiation codon for the sapA2gene.The asterisk indicates the stopcodon.Putative Shine-Delgarno(SD)and chi sequences(Ϫ13toϪ9andϪ38toϪ31,respectively)upstream of the initiator methionine,as well as the penta-meric repeat(ATTTT;Ϫ31toϪ17),are indicated.Converging arrows indicateinverted repeat sequences ending at the ATG initiation codon and3Јto theORF.The DNA sequence beginning at base3336may form a dual stem structure(⌬GϭϪ19.4).A10-bp TTTTAAATTT motif is repeatedfive times within thedownstream noncoding region(boldface in boxes),as are nearly identical se-quences(boldface only).Repeat pairs(A and B)show additionalflanking se-quence similarity or identity.1736DWORKIN ET AL.J.B ACTERIOL.cloned from C.fetus 82-40LP3was identical to the partial sequence located downstream of sapA1which was cloned from the unrelated C.fetus strain 23B (47);this is consistent with the plasmid restriction maps (see Fig.4).These findings sug-gested that sapA1and sapA2exist in tandem on the chromo-some and that this property is highly conserved among C.fetus parison with the two previously cloned sapA ho-mologs demonstrated two regions of identity.The first began 74bp before the ORF,proceeding 552bp into the ORF.Within this shared region,a sequence (GCTGGTGA)shar-ing seven of eight bases with the E.coli RecBCD (Chi)recog-nition site (GCTGGTGG)(12,13)was found upstream of the ATG start codon and was followed by three pentameric (AT-TTT)repeats and a potential ribosome-binding site (AGGAG;Fig.2).A perfect 7-bp palindrome with a 1-bp spacer ending exactly at the ATG initiation codon and encompassing the ribosome-binding site also was present (Fig.2).The second region of identity (Fig.3)began 6nucleotides from the sapA2translation stop codon and extended for 51bases,with subse-quent brief areas of homology over the next 50bases.A puta-tive transcriptional termination signal with a ⌬G of Ϫ19.4also was observed.Within the downstream noncoding region,a 10-bp motif (TTTTAAATTT)was repeated five times,and nearly identical motifs also were present (Fig.2).The first repeat commenced at the 3Јend of the 50-to 60-bp conserved region shared among all sapA homologs (Fig.3).From there it was repeated a variable number of times downstream of each sapA homolog (not shown).Sequence flanking the re-peat also may have been duplicated (Fig.2).Within the down-stream conserved region,an 8-bp palindromic sequence similar to that found immediately upstream of the initiation codon was observed (Fig.2and 3).The presence of these homol-ogous sequences at both the 5Јends of these sapA homologs and 3Јto them suggested that these regions might play a role in homologous recombination,as previously reported (47).Sequence analysis does not demonstrate the presence of a typical ␴70-like promoter (utilized by at least one S-layer pro-tein expressed in C.fetus [46])or any other known promoter upstream of sapA2(Fig.2).Northern blot analysis using a unique sapA2-specific 0.46-kb Pst I fragment as probe (probe 6[Fig.4])indicated that sapA2was not expressed in strain 82-40LP3,from which it was cloned (data not shown).However,for the laboratory-passed strain 23D,which represents a mixed culture expressing both 97-and 127-kDa S-layer proteins,and a laboratory variant created in a manner similar to that de-scribed elsewhere (47),the sapA2-specific probe hybridized to 3.3-kb messages in both (data not shown).Thus,sapA2can in fact be expressed by C.fetus .These results indicated that,consistent with the lack of an upstream promoter-like se-quence,sapA2represents a silent gene in the strain (82-40LP3)from which it was cloned but is actively expressed in other strains producing 127-kDa S-layer proteins.Comparison of predicted primary amino acid parison of sapA2with sapA or sapA1demonstrated an identical N-terminal 184-amino-acid sequence (Fig.4).For the remainder of the ORF,sapA2shared 28%identity (47.7%similarity)and 26%identity (48.8%similarity)with sapA and sapA1,respectively.sapA2and sapA shared an identical span of eight residues (starting at position 234);no other significant regions of identity were found between sapA2and sapA or sapA1.In contrast,comparison between sapA and sapA1be-yond residue 184demonstrated a 45.4%identity (64.8%sim-ilarity)with extension of the N-terminal region of identity to residue 219and five identical clusters of identity of six or more amino acids in the remainder of the molecules.Thus,sapA and sapA1were more closely related to each other than to sapA2.PFGE.Given the tandem nature of the two sapA homologs,we sought to determine whether the family of sapA homologs (4,46,47)are clustered on the chromosome.When chromo-somal DNAs from five C.fetus strains were digested with the infrequent cutter Sal I and resolved by PFGE (Fig.5,left pan-el),the fragmentation pattern was nearly identical for all.Sub-sequent Southern blotting (Fig.5,right panel)with the con-served 5Јregion of the sapA homologs as a probe (probe 1[Fig.4])demonstrated a limited hybridization pattern.Strain 23D showed a hybridizing band of 125kb,whereas its spontaneous S Ϫmutant 23B showed a hybridizing fragment at 116kb.Strain 82-40LP and its two spontaneous variants,82-40HP and 82-40LP3,all showed two hybridizing signals;a 54-and 81-kb band were seen for strains 82-40LP and 82-40LP3,whereas 82-40HP possessed two hybridizing signals at 54and 73kb.Consis-tent findings also were found for Sac II,Bgl I,and Mlu I digests of C.fetus 23D;Sac II digestion demonstrated a unique hybrid-izing band of 93kDa,Bgl I digestion demonstrated three hy-bridizing bands totalling less than 130kb,and Mlu Idigestionparison of sapA ,sapA1,and sapA2downstream nucleotide se-quences.Converging arrows indicate inverted repeat sequences within a region of strong homology.Colons indicate identical nucleotides.Numbers represent nucleotide positions in relation to the translation initiation codon (ATG)for sapA homologs.The boldface indicates a 10-bp repeat as in Fig.2.FIG.4.Physical map comparisons of regions surrounding sapA homologs.Mapping was performed in pBG1and pBG101for sapA ,pMT101for sapA1,and pMJ1for sapA2.Arrows represent the locations of genes and directions of transcription.Hatched boxes indicate the upstream and N terminus-encoding conserved regions.The smaller black boxes indicate homologous regions down-stream of each ORF as indicated in Fig.3.The locations of DNA probes used for Southern and Northern hybridizations are indicated by the numbers 0to 6.0,0.5-kb Cla I-Nde I fragment of the sapA promoter region;1,0.42-kb PCR frag-ment of conserved origin;2,0.36-kb PCR fragment proximal to the N terminus;3,0.35-kb Hin dIII fragment of the middle region;4,0.46Hin dIII fragment of the middle region;5,0.33-kb Pst I fragment of the C-terminal region;6,0.46-kb Pst I fragment of the C-terminal region.Restriction endonuclease cleavage sites:A,Acc I;B,Bgl II;B1,Bst nI;C,Cla I;E,Eco RI;H,Hin dIII;H2,Hin cII;N,Nde I;Na1,Nal I;P,Pst I;P2,Pvu II;S,Ssp I;X3,Xma III.V OL .177,1995LPS-BINDING DOMAIN OF C.FETUS S-LAYER PROTEIN 1737demonstrated two hybridizing bands totalling less than 95kb (data not shown).Because the hybridization patterns were limited to one or two restriction fragments totalling as little as 93kb,these results indicated that the sapA homologs were clustered on fragments representing less than 8%of the chro-mosome (37).The spontaneous mutants 23B and 82-40HP appeared to differ from their wild-type parents by deletion of approximately 8to 9kb within this region.Reblotting using the sapA promoter region (probe 0[Fig.4])demonstrated a single Sal I-hybridizing band in S ϩstrains at 125kb in strain 23D and 81kb in strains 82-40LP and 82-40LP3but no band for 82-40HP or 23B,as expected (46)(not shown).Southern analysis with sapA2probes.To investigate the genomic organization of sapA2within the strain from which it was cloned (82-40LP3)and within other C.fetus strains that produce smaller S-layer proteins,a series of Southern hybrid-izations were performed with probes 1to 6from pMJ1(Fig.4).As shown in Fig.6,probes increasingly 3Јwithin the ORF hybridized to progressively fewer Hae III fragments.The N-terminally conserved probe (probe 1)hybrized to multiple (six)bands,some of which appeared as doublets,in all strains (Fig.6A,left panel),although fewer hybridizing bands were re-solved in the spontaneous S Ϫmutant strains 23B and 82-40HP.Probe 2,which flanks the conserved N-terminal region at its 3Јend,hybridized strongly to three bands and weakly to one (23D)or two (LP,HP,and LP3)bands (Fig.6A,middle band).A strongly hybridizing band of approximately 2.0kb in the other four strains was absent in strain 23B (Fig.6A and B).A highly conserved 4.3-kb Hae III fragment containing sapA2se-quences was present,since probes 2to 6recognized it in all strains (Fig.6A and B).Probe 3from the middle region of sapA2hybridized to the sapA24.3-kb fragment as well as two homologs of 2.0and 1.0kb,with the exception of 23B (Fig.6A,right panel).Only two hybridization signals at 4.3and 2.0kb were observed with probes 4and 5(Fig.6B,middle and left panels).In all strains examined,probe 6from the extreme C-terminus-encoding region of the sapA2ORF hybridized only to the 4.3-kb sapA2fragment (Fig.6A and B).Consistent results were also observed for Acc I-,Bgl II-,and Hin dIII-di-gested genomic DNAs,since sapA2probes increasingly 3Јhy-bridized to progressively fewer fragments (data not shown).Taken together,these results demonstrated that sapA2existed as a complete copy within the genome of each of the C.fetus strains examined and that homology between sapA2and other sapA homologs diminished toward the 3Јend of the gene.These data also clearly demonstrated that the deletions ob-served in strains 23B and 82-40LP were different.Reattachment assays with truncated products.S-layer pro-teins of C.fetus exclusively adhere to the LPS layer in a sero-type-specific manner (53).On the basis of recombinant pro-teins reattaching to the surface of S ϪC.fetus mutants,the LPS-binding domain of the 97-kDa S-layer protein has been shown to be present within the N-terminal half of the molecule (53).Because the three type A S-layer proteins that have been examined have been deduced to share the same 184N-termi-nal amino acids and then diverge,it was anticipated that this 5Јregion encoded the type A binding domain.To verify this hypothesis,we performed reattachment assays using E.coli -expressed cloned full-length and truncated sapA2recombinant products,derived from exonuclease III-generated nested de-letions.The full-length sapA2recombinant S-layer protein re-attached to type A cells but not to type B cells (Fig.7),as expected (53),demonstrating that the product of sapA2,hith-erto unexpressed in C.fetus ,possessed functional binding ca-pacity that was specific for type A LPS.Both the 40(C16-4)-and 23(C17)-kDa deletion mutants also reattached to type A but not to type B S Ϫcells (Fig.7).As determined by dideoxy DNA sequencing,the mutant C17was truncated at amino acid residue 189.Thus,the conserved 189-amino-acid N-terminal peptide contained at least one of the LPS type-specific binding sites.More extensively truncated recombinant proteins were not sufficiently immunoreactive to be detectable by immuno-blotting so as to narrow further the LPS-bindingdomain.FIG.5.PFGE and Southern hybridization using probe 1of genomic DNA from C.fetus 23D,23B,82-40LP,82-40HP,and 82-40LP3.Genomic DNAs were digested overnight with 60U of Sal I while embedded in an agar plug.Digested material was electrophoresed with a CHEF system for 24h at 185V and 8ЊC,with a ramped pulse time of 10to 25s in a 1%agarose gel.The gel was photographed (left panel)and transferred to nylon membrane for Southern hybridization with probe 1(right panel).Molecular size (MW)markers are inkilobases.FIG.6.Southern hybridization with six sapA2probes to genomic DNAs from C.fetus 23D,23B,82-40LP,82-40HP,and 82-40LP3.Chromosomal DNAs from these strains were digested with Hae III;the restriction fragments were separated an a 0.7%agarose gel,transferred to a nylon membrane,and hybrid-ized with the 32P-labeled sapA2probes indicated in Fig.4(probes 1through 3[A]and probes 4through 6[B]).The positions of molecular size markers (in kilo-bases)are indicated to the left of each panel.1738DWORKIN ET AL.J.B ACTERIOL .。